Choosing a Format


In what formats can the shRNA clones be ordered?
Sigma-Aldrich is both a collaborative member and supplier of TRC1 and TRC2. The TRC1 library currently contains more than 159,000 pre-cloned shRNA constructs targeting 16,000 annotated human genes and 15,950 annotated mouse genes. The TRC2 library contains more than 150,000 pre-cloned shRNA constructs. Most genes have at least 3 individual clones from which to choose. Three formats are available: bacterial glycerol stock, purified plasmid DNA, and lentiviral particles. For more information on ordering MISSION® products, please click here.


If I want to make my own viral particles, which format should I purchase?
Sigma offers ready-to-use viral particles to eliminate these steps for you. However, if you choose to make your own viral particles, we recommend the purified plasmid format for packaging protocols that require less than 1 µg of plasmid DNA (i.e. typical protocols for 60 mm dishes utilize 1 µg of transfer vector and yield 4-5ml of viral particles). For large scale production of viral particles, we recommend the bacterial glycerol stocks. The bacterial format allows for propagation of the shRNA transfer vector and facilitates larger scale DNA purification required for scaled up packaging co-transfections. Our Lentiviral Packaging Mix is an optimized formulation for generating lentivirus in HEK293T cells.

Why would I use the purified plasmid DNA format?
The DNA format (1 µg) is ideal for doing small transfections in 96-well plates (with replicates) or for small-scale packaging co-tranfections. Plasmid purification is a costly and time-consuming step that is eliminated with this product format.

How many cells can I infect with the amount of lentivirus provided?
The amount of cells that can be infected depends upon the cell line being used. For HeLa, A549 and HEK293T cells we have used 4µL (1 x 106 TU/ml via p24 assay) for each well containing ~16,000 cells in a 96-well plate. Primary or other difficult cells may require more lentiviral supernatant, but even at a 10 µL level, it would give you enough for 20 x 96-well reactions. We suggest decreasing the number of cells plated to increase the multiplicity of infection (MOI) if necessary. Also, performing a limiting dilution titer on your cell line will give you the optimal amount of viral particles needed for each assay.

Can I extract the vector directly from the lentiviral particle?
No, this is not possible. The vector is used to make the particles in the packaging or producer cells. The viral genome contains only the RNA version of the region found between the 5’ and 3’ LTRs of pLKO.1 (promoter, hairpin sequence, puromycin resistance gene, etc.).

Can individual MISSION TRC shRNA constructs be purchased and in what format are they available?
Individual clones may be purchased in any of the three available formats: bacterial stock, purified DNA and lentiviral transduction particles.

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